1. Field of the Invention
This invention provides for superior nucleic acid primers for amplification of select target regions of the genome of the genus Legionella. The invention facilitates detection of pathogenic and nonpathogenic forms of this genus. The invention further provides for processes for using the primers in template dependent nucleic acid polymerase extension reactions to amplify select target regions. Kits for the use of these primers are also provided.
This invention further provides for methods of controlling the intensity of visual signal for detection of duplex formation in nucleic acid hybridization assays under high stringent conditions. This method involves the blending of different capture probes onto a solid support.
2. Information Disclosure
Legionella species are known as both pathogenic and nonpathogenic microorganisms. In the pneumonic form, they are intracellular pathogens of lung macrophage cells. Legionellaceae, Chpt. 9260 J. in Standard Methods of the Examination of Water and Wastewater, Eds. Clesceri, Greenberg and Trussell, 17th Ed. 1989 pages 9-149 to 9-153 and Muraca, P. W. et al., 1988, Environmental Aspects of Legionnaires' Disease, J. Amer. Water Works Assoc. 80:78-86.
A surface antigen of Legionella has been implicated as a requirement for intracellular pathogenicity and is called a macrophage infectivity potentiator or mip. Cianciotto, N. P. et al., 1989, A Legionella pneumophila Gene Encoding a Species-specific Surface Protein Potentiates Initiation of Intracellular Infection, Infection and Immunity, 57:1255-1262.
The nucleotide sequence of the 5S rRNA has been reported by MacDonnell, M. T. and R. R. Colwell, 1987, The Nucleotide Sequence of the 5S rRNA From Legionella pneumophila, Nucleic Acid Research, 15:1335; and, by Chumakov, K. M et al., 1986, Use of 5S Ribosomal RNA Nucleotide Sequence Analysis for the Study of Phylogeny of the Genus Legionella, Mol. Genet., Mikrobiol Virusol., 8:38-40.
The nucleotide and amino acid sequence of the macrophage infectivity protein is known and reported by Engleberg, N. C. et al., 1989, DNA Sequence of mip, a Legionella pneumophila Gene Associated With Macrophage Infectivity, Infection and Immunity, 57:1263-1270.
Standard culture techniques for Legionella are not adequate to properly assess risks from this deadly patbogen. Hussong, D. et al, 1987, Viable Legionella pneumophila Not Detectable by Culture on Agar Media, Bio/Technology 5:947-950.
Nucleic acid probes for detection of Legionella pneumophila have been reported. Grimont, P. A. D. et al., 1985, DNA Probe Specific for Legionella pneumophila, J. Clin. Micro., 21:431-437; and Engleberg, N. C. et al., 1986, A Legionella-Specific DNA Probe Detects Organisms in Lung Tissue Homogenates from Intranasally Inoculated Mice, Israel J. of Med. Sciences, 22:703-705.
The use of polymerase chain reaction amplification methods to detect Legionella species has been disclosed by Starnbach, M. N. et al., 1989, Species-Specific Detection of Legionella pneumophila in Water by DNA Amplification and Hybridization, J. of Clin. Microbiol., 27:1257-1261; in U.S. Ser. No. 07/467,813 filed on Jan. 1, 1990; and, also in Detection of Legionella in Environmental Samples Using Polymerase Chain Reaction (PCR), Biotechnology Bulletin, May 1990 pages 11-12.